Our long-term objectives are to define mechanisms how neurotransmitter receptors contribute to neonatal hypoxic-ischemic (HI) brain injury, and to search for novel and specific therapeutic targets for neonatal HI. Our hypothesis for this project is: Fyn contributes to neonatal HI brain injury through regulation of both synaptic and extrasynaptic NR2B signaling pathways Aim 1: Determine how Fyn regulates NR2B complex composition at different postsynaptic locations after neonatal HI. Hypothesis: Synaptic and extrasynaptic NMDAR assemble different protein complexes in normal brain and after HI, which are modified by Fyn. 1A) Purify synaptic and extrasynaptic membranes from sham and HI-injured WT, Fyn overexpressing mice, PP2-treated mice and Y1472 knock-in mice. 1B) Use IP to pull down NR2B complexes from the two compartments. 1C) Use proteomic analysis to identify synaptic and extrasynaptic NR2B binding partners. 1D) Validate synaptic and extrasynaptic NR2B binding partners. Aim 2: Assess the functional significance of Fyn phosphorylation of NR2B at Y1472 after neonatal HI Hypothesis: Fyn phosphorylation of NR2B at Y1472 mediates HI brain injury through upregulation of superoxide and nitric oxide (NO) formation 2A) Assess brain injury in Y1472 knock-in mice following HI. 2B) Investigate the mechanisms by which NR2B phosphorylation at Y1472 regulates superoxide and NO formation after HI. 2C) Determine if Y1472 affects NR2B expression and synaptic/extrasynaptic localization. 2D) Determine if Y1472 affects NR2B association with downstream signaling proteins. Aim 3: Determine the individual function of Fyn-mediated NR2B phosphorylation site and the coordination of three sites following HI in vitro. Hypothesis: Fyn phosphorylation of specific residues on the NR2B activates either survival or cell death signaling pathways. 3A). Use the LEMPRA (lentiviral-mediated protein replacement assay) constructs to knockdown endogenous NR2B and simultaneously express exogenous NR2B with single (Y1472, Y1252 or Y1336), double or triple phosphorylation mutants in primary cortical neurons. 3B). Measure cell death and NMDAR surface expression in neurons with LEMPRA constructs following oxygen glucose deprivation or NMDA exposure. 3C). Perform electrophysiology and calcium imaging for cells with LEMPRA constructs as functional readouts of NMDAR activity. Brain hypoxia-ischemia is a leading cause of neonatal mortality and disabilities in children. This proposal represents the first systematic and in-depth examination of how Fyn-mediated NR2B phosphorylation is involved in the pathogenesis of HI brain injury in the newborn. Moreover, identification of location-dependent NR2B binding partners has not been characterized in the developing brain, or how it changes in response to HI. This work will inform novel therapeutic approaches to selectively uncouple the NMDAR or Fyn from harmful consequences while preserving their important beneficial functions in neonates.